According to the World Health Organization, 33.3 million people worldwide were living with Human Immunodeficiency Virus (“HIV”) as of 2009. That same year, 2.6 million new infections were reported and 1.8 million people worldwide died of AIDS-related illnesses.
Human immunodeficiency virus type 1 (HIV-1) enters host cells through a multistep process that requires sequential interactions of the envelope glycoprotein gp120. The envelope protein interacts first with the CD4 receptor and then with one of a family of chemokine coreceptors, mainly CCR5 or CXCR4. The V3 loop in HIV-1 gp120 has been shown to be critical for coreceptor binding.
Importantly, HIV-1 strains can be phenotypically classified according to a virus' ability to use the CCR5 (R5) and/or CXCR4 (X4) co-receptor. Pure R5-tropic and pure X4-tropic virus can use only the CCR5 and CXCR4 co-receptors to enter the target cell, respectively, while dual-tropic virus can use both co-receptors. In a virus population, the use of both co-receptors can be due either to the presence of dual-tropic clones or to a mixture of pure R5-tropic and X4-tropic clones or both. This is cumulatively defined as dual/mixed phenotype.
HIV-1 co-receptor usage is of central pathological and clinical importance. It has been shown that R5-tropic viruses are generally responsible for the establishment of the initial infection and predominate in the majority of newly HIV-1 infected patients, while the use of the CXCR4 co-receptor is generally seen in more advanced stages of disease, and has been associated with a more rapid CD4 decline and progression to AIDS.
HIV-1 co-receptor usage is also of critical therapeutic importance given the current and future approval of CCR5 antagonists for the treatment of HIV-1 infection. CCR5-antagonists are a new class of anti-HIV-1 drugs that specifically inhibit the entry of R5-tropic HIV-1 strains into the target cells by allosteric inhibition of the CCR5 co-receptor. Maraviroc is the first approved CCR5 antagonist, which entered clinical practice in 2007.
Significantly, HIV-1 tropism must be determined before CCR5 antagonists can be prescribed. CCR5-antagonists are most effective (and the viruses less likely to develop resistance) when administered to treatment-naïve patients. This is due to the higher prevalence of CCR5-tropic HIV-1 early in the infection cycle and relative to more advanced patients. Moreover, CCR5 antagonists are ineffective against X4-tropic viruses.
The mandatory determination of HIV-1 tropism prior to administration of CCR5 antagonists presents several practical difficulties. A number of phenotypic assays have been developed, such as the Trofile® assay (Monogram Biosciences). Trofile® is a single-cycle recombinant virus assay in which a pseudovirus is generated from full length envelope (env) genes derived from the patient's virus population. The complete envelope is used to determine viral tropism taking into account determinants that lie outside the V3 loop. However, phenotypic assays are complex and marked by high cost and long turnaround times. In addition, most of them cannot determine HIV-1 tropism in clinical samples with viral loads below 1,000 copies/ml and have an unacceptably high sensitivity threshold for detection of the minority X4-tropic virus population. Thus, a need exists for genotypic tropism assays that can rapidly and efficiently determine tropism based on the sequence of a patient-derived V3 loop in HIV-1 gp120.
In addition to these genotyping needs, there is a long-felt need for accurate quantification of HIV-1 RNA and proviral DNA. RNA quantification, also referred to as viral load measurements, are routinely used in clinical settings. (FIG. 1). Indeed, studies have shown HIV-1 RNA levels to be a predictor of the time to progression to acquired immunodeficiency syndrome (AIDS) and death that is independent of CD4 cell counts. Viral load measurements are also useful in determining when to initiate anti-retroviral therapy and in monitoring the response to such therapy. In specific situations, HIV-1 RNA and proviral DNA levels may also be useful in diagnosing of HIV infection. For example, serologic testing may not reliably identify HIV-1 infection in neonates with passively acquired maternal HIV-1 antibodies or with incompletely developed immune systems, in individuals with early infection (<30 days from infection), or with “indeterminate” antibody profiles by Western blot assays. In these situations, detection and quantification of HIV-1 nucleic acids (RNA or proviral DNA) can provide early evidence of HIV-1 infection (approximately 10-14 days after infection) and can also provide early evaluation of the progression of infection.